Cloning And Functional Analysis Of DREB Transcription Factors In Nictotiana Benthamiana

Drought, high-salinity and freezing are the major environmental factors that influence the development, growth and quality of plant. In the agricultural production of tobacco, low-temperature after being transplanted and drought in seedbed and later phase of tobacco growth, which influence the development, growth and quality forming of plant, bring the tobacco grower irreversible economic loss. To solve these problems, tobacco scientists took many measures of cultural and management in field for long time, but very little effect has been acquired on them. In order to ravel out them absolutely, we should rebuild the adaptability and stress-tolerant of tobacco from molecular level, which is the only method. Isolation, cloning and functional analysis the DREB transcription factors in tobacco, which could control a series of genes expression that resist abiotic conditions, should provide the foundation for improving the tobacco stress-tolerance by using molecular biology methods. In this study, a wild tobacco Nicotiana Benthamiana was used experiment material. Two DREB transcription factors were cloned and functional analyzed, and a key residue amino acid was found to affect the DREBs binding to DRE cis-elements in tobacco. The main results are as following:1. Analyzing the EST sequences from GenBank, we obtained two DREB-like genes from Nicotiana Benthamiana using biotech methods and named NbDREB1 and NbDREB2, which possess typical structural characteristics of AP2/EREBP domain. Then the two genes was constructed into yeast expression vector YepGAP, and the yeast carrying the WDRE could neither grow on plate of SD/His~- Ura~- Tip~- + 0.03 mol/L 3-AT, nor activate the reporter gene. Furthermore, NbDREB1 and NbDREB2 were cloned into pGADT7, Yeast in vivo analysis showed that on a selective medium plate of SD/His~- Ura~- Trp~- +0.03 mol/L 3-AT, and induced LacZ activity only in the transformation of NbDREB1. The results show that NbDREB1 could recognize and bind the cis-element DRE, and maybe an inactivity DREB transcription factor.